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Design of New Selenium Enzymes and Proteins, and Their Functions

Research Project

Project/Area Number 02454545
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 代謝生物化学
Research InstitutionKyoto University

Principal Investigator

SODA Kenji  Institute for Chemical Research, Kyoto University, Professor, 化学研究所, 教授 (30027023)

Co-Investigator(Kenkyū-buntansha) HIRASAWA Toshiko  The same institution and department Technical Associate, 化学研究所, 教務職員
YOKOIGAWA Kumio  The same institution and department Research Associate, 化学研究所, 助手 (60230637)
ESAKI Nobuyoshi  The same institution and department Associate Professor, 化学研究所, 助教授 (50135597)
吉村 徹  京都大学化学研究所, 助手 (70182821)
Project Period (FY) 1990 – 1991
Project Status Completed (Fiscal Year 1991)
Budget Amount *help
¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1991: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1990: ¥3,400,000 (Direct Cost: ¥3,400,000)
KeywordsSelenium / Selenocysteine / Selenomethionine / Selenium Proteins / Peptide Synthesis / Opal Stop Codon / Metallothionein / Glutathione
Research Abstract

The chromosomal DNA of mouse GSHPx gene was reconstructed to express in E. coli. It contains a single intron within the structural gene. Then, BamHI sites were introduced at both the ends of the intron by oligonucleotide-directed mutagenesis, and the following digestion with BamHI and ligation eliminated the intron. The reconstructed gene was subcloned into the E. coli expression vector, pKK223-3, and E. coli JM109 cells were transformed with the resulting plasmid. The transfomant was incubated with ^<35>S-methionine and ^<75>Se-Na_2SeO_3 to label the proteins. However, GSHPx was not synthesized under the conditions used.
The gene fragments of GSHPx which contain the TGA codon encoding the SeCys residue were fused with the gene of beta-gal. When the TGA is readthrough, the fused protein most probably has beta-gal activity, which is easily detected with X-Gal. The transformants showed beta-gal activity only when cultured anaerobically. The fused proteins produced under anaerobic conditio … More ns were labeled with ^<75>Se-Na_2SeO_3. These results suggest that some factors suppressing the nonsense codon TGA are formed under anaerobic conditions, and consequently selenium is incorporated into the fused protein.
We synthesized a selenocysteine-containing peptide, metalloselenonein, which contains selenocysteine residues substituted for all cysteine residues in Neurospora crassa copper metallothionein, using an automated peptide synthesizer. Metalloselenonein binds 3 gram atoms of Cu (I) per mol. This adduct showed a broad absorption band between 230 and 400 nm and a fluorescence band at 395 nm, which can be attributed to copper-selenolate coordination. The circular dichroism spectrum of the Cu-metalloselenonein complex showed a positive CD band around 245 nm attributable to asymmetry in metal coordination. The Cu-bound metalloselenonein was analyzed by Cu-and Se-EXAFS. the results revealed that three different Cu-Se bonds occur in Cu-SeMT, and the ratio of occurrence was 3, 1, and 3, respectively. Less

Report

(3 results)
  • 1991 Annual Research Report   Final Research Report Summary
  • 1990 Annual Research Report
  • Research Products

    (8 results)

All Other

All Publications (8 results)

  • [Publications] Tadao Oikawa: "Metalloselenonein,the Selenium Analogue of Metallothionein:Synthesis and Characterization of Its Complex with Copper Ions." Proceedings of National Academy of Science,U.S.A.88. 3057-3059 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Tadao Oikawa: "Synthesis and Characterization of a Selenium Analogue of Glutathionine,Glutaselenone" Analytical Biochemistry.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Tadao Oikawa: "Metalloselenonein, The Selenium Analogue of Metallothionein Synthesis and Characterization of Its Complex with Copper Ions" Proceedings of National Academy of Science, U. S. A.88. 3057-3059 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Tadao Oikawa: "Synthesis and Characterization of a Selenium Analogue of Glutathione, Glutaselenone" Analytical Biochemistry.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Tadao Oikawa: "Metalloselenonein,the Selenium Analogue of Metallothionein: Synthesis and Characterization of Its Complex with Copper Ions." Proceedings of National Academy of Science,U.S.A.88. 3057-3059 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Tadao Oikawa: "Synthesis and Characterization of a Selenium Analogue of Glutathionine,Gluteselenone" Analytical Biochemistry.

    • Related Report
      1991 Annual Research Report
  • [Publications] Tadao Oikawa: "Metalloselenonein,the Selenium Analogue of Metallothionine:Synthesis and Characterization of Its Copper Complex with Copper Ions" Proceedings of National Academy of Science,U.S.A.

    • Related Report
      1990 Annual Research Report
  • [Publications] Tadao Oikawa: "Synthesis and Characterization of a Selenium Analogue of Glutathionine,Glutaselenone" Analytical Biochemistry.

    • Related Report
      1990 Annual Research Report

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Published: 1990-04-01   Modified: 2016-04-21  

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